Biomedical subjects

R Lahesmaa

Publications and source records attributed to R Lahesmaa.

At least 19 recordsLinked to original sources

PubMed2006

Intranasal administration of chlamydial outer protein N (CopN) induces protection against pulmonary Chlamydia pneumoniae infection in a mouse model.

Chlamydia pneumoniae is an intracellular pathogen that grows inside a vacuole, referred to as an inclusion. C. pneumoniae possess a type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and to the host cell cytosol. Proteins such as chlamydial outer protein N (CopN) that associate with the inclusion membrane are potential targets for the host's MHC-dependent antigen presentation, thereby representing ideal antigen candidates for T cell-based vaccination. The results of this study showed that intranasal immunization of BALB/c mice with heat-aggregated CopN protein and an Escherichia coli heat-labile toxin (LT) induced a strong immune response, detected as antigen-specific antibody production, lymphocyte proliferation and IFN-gamma production. Furthermore, the immunization induced statistically significant protection against intranasal C. pneumoniae challenge, the level of which correlated with the magnitude of CopN-specific lymphocyte proliferation. Both heat-aggregation of the antigen and the presence of LT adjuvant were required for maximal protective effect.

Administration, Intranasal↗

PubMed2005

Global profiling of coxsackievirus- and cytokine-induced gene expression in human pancreatic islets.

AIMS/HYPOTHESIS: It is thought that enterovirus infections initiate or facilitate the pathogenetic processes leading to type 1 diabetes. Exposure of cultured human islets to cytolytic enterovirus strains kills beta cells after a protracted period, suggesting a role for secondary virus-induced factors such as cytokines. METHODS: To clarify the molecular mechanisms involved in virus-induced beta cell destruction, we analysed the global pattern of gene expression in human islets. After 48 h, RNA was extracted from three independent human islet preparations infected with coxsackievirus B5 or exposed to interleukin 1beta (50 U/ml) plus interferon gamma (1,000 U/ml), and gene expression profiles were analysed using Affymetrix HG-U133A gene chips, which enable simultaneous analysis of 22,000 probe sets. RESULTS: As many as 13,077 genes were detected in control human islets, and 945 and 1293 single genes were found to be modified by exposure to viral infection and the indicated cytokines, respectively. Four hundred and eighty-four genes were similarly modified by the cytokines and viral infection. CONCLUSIONS/INTERPRETATION: The large number of modified genes observed emphasises the complex responses of human islet cells to agents potentially involved in insulitis. Notably, both cytokines and viral infection significantly (p<0.02) increased the expression of several chemokines, the cytokine IL-15 and the intercellular adhesion molecule ICAM-1, which might contribute to the homing and activation of mononuclear cells in the islets during infection and/or an early autoimmune response. The present results provide novel insights into the molecular mechanisms involved in viral- and cytokine-induced human beta cell dysfunction and death.

Aged↗

PubMed2005

A human ImmunoChip cDNA microarray provides a comprehensive tool to study immune responses.

DNA microarray technology has developed rapidly in recent years and has become an essential tool, providing novel approaches to biomedical research. In this paper, we describe a self-designed ImmunoChip cDNA array for immunological research. With a comprehensive selection of genes of interest, we can focus on key signalling pathways and molecular mechanisms at relatively low cost compared to commercial platforms which are usually targeted at global screening of gene expression. To validate the efficiency of the ImmunoChip, we studied T helper cell polarization to functionally distinct subsets (Th1 and Th2). We also developed a tool for quality control of cDNA microarrays that assesses the technical quality of an ImmunoChip. The information produced with the quality control tool is shown to be valuable for extracting correct information from cDNA microarrays. Gene expression measurements with ImmunoChip are in agreement with the results obtained using oligonucleotide microarrays and with published quantitative RT-PCR data. The ImmunoChip provides reliable measurements and gives new insights into various aspects of human immune responses.

DNA, Complementary↗

PubMed2005

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants.

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.

Biopsy↗

PubMed2005

Clearance of Chlamydia pneumoniae infection in H-2 class I human leucocyte antigen-A2.1 monochain transgenic mice.

CD8+ T cells have been suggested to play an important role in protective immunity against pulmonary Chlamydia pneumoniae infection in mice. Moreover, several classical major histocompatibility complex class I - restricted cytotoxic CD8+ T lymphocytes (CTL) specific for C. pneumoniae- derived peptides have been identified. Here, we studied the outcome of C. pneumoniae infection in human leucocyte antigen (HLA)-A2.1 transgenic mice (HHD mice) that are only able to express a classical human class I molecule (HLA-A2.1). C. pneumoniae infection was self-restricted in HHD mice which were able to develop specific immune responses and a protective immunity against a subsequent rechallenge in a manner comparable to wildtype mice. Furthermore, accumulation of functional and C. pneumoniae-specific T cells to the site of infection was detected after challenge. Antigen processing and HLA-A2.1-dependent presentation was studied by immunizing the HHD mice with chlamydial outer protein N (CopN). Isolation of a peptide-specific CTL line from the CopN-immunized mice suggests that the HLA-A2.1 molecule can support the development of CTL response against a chlamydial protein in mice. These findings suggest that the transgenic mouse model can be used for further characterization of the HLA-A2.1-restricted CD8+ T-cell response during C. pneumoniae infection and for identification of CD8 epitopes from chlamydial antigens.

Animals↗

PubMed2004

Association study of 15 novel single-nucleotide polymorphisms of the T-bet locus among Finnish asthma families.

OBJECTIVE: T-box expressed in T cells (T-bet) is a transcription factor regulating the commitment of T helper (Th) cells by driving the cells into the Th1 direction. Abnormal Th1/Th2 balance may lead to complex disorders like asthma or autoimmune diseases. Recent studies have suggested that T-bet might be a candidate gene for asthma. This led us to screen 23 Finnish individuals for single-nucleotide polymorphisms (SNPs) in the T-bet locus and study the association between the SNPs and high serum IgE level and asthma. METHODS: We screened all six exons, adjacent intronic areas and 2 kb of the 5'-flanking region from 23 individuals utilizing WAVE trade mark technology. To explore whether T-bet is associated in serum IgE regulation or asthma we genotyped the SNPs in a Finnish asthmatic founder population. The association analyses were made using haplotype pattern mining. RESULTS: Fifteen novel SNPs were found in the T-bet gene. Within the Finnish asthmatic founder population, there was no association between T-bet SNPs and high serum IgE level or asthma. CONCLUSIONS: The genetic variability in the T-bet gene does not play a role in the pathogenesis of human asthma. Our results provide a novel panel of SNPs in T-bet and will help determine whether the SNPs have a functional role in other T cell-mediated diseases.

Asthma↗

PubMed2003

Downregulation of mafB expression in T-helper cells during early differentiation in vitro.

We have studied the expression of a human homologue of mafB (maf-1), a member of the family of large maf transcription factors. In support of the suggested key role that mafB expression plays in differentiating macrophages, we found mafB to be expressed at a very high level in monocytic U937 and THP-1 cell lines. However, we show here that mafB transcription is not restricted to myeloid cells but can also be detected in lymphoid cells, indicating transcriptional plasticity during haematopoiesis. In conclusion, strong proliferative signals mediated by T-cell activation and interleukins (IL-4 and IL-12) downregulate the mafB messenger RNA transcript level when resting naïve CD4+ T-helper cells enter the differentiation pathway in vitro.

Avian Proteins↗

PubMed2001

Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR.

Real-time RT-PCR method was exploited to identify endogenous reference genes in differentiating human T helper cells. When using this technology in our experimental system, finding a set of genes whose mRNA expression levels would not change appeared to be very challenging. Our initial plan to use the expression level of GAPDH in normalizing the results failed, because the mRNA expression of GAPDH underwent significant changes during the cell culture. Additional studies on the transcription of several other classical housekeeping genes led to similar results. Our second approach was to use results from an extensive survey of gene expression done by oligonucleotide microarrays and to select another panel of genes for testing. This resulted in the identification of three genes whose expression was relatively stable in our experimental system and, therefore, suitable as endogenous reference genes in these cells. The results indicate that the expression level of a constitutively expressed gene may change during the cell culture in vitro, which emphasizes again the importance of carefully validating endogenous control genes for comparative quantification.

Cell Differentiation↗

PubMed2001

Distinct gene expression profiles of human type 1 and type 2 T helper cells.

BACKGROUND: The development and activation of CD4+ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4+ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method. RESULTS: In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.

CD4 Antigens↗

PubMed2001

A proteome database of human primary T helper cells.

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.

Databases, Protein↗

PubMed2000

Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 cells.

We have used a real-time quantitative RT-PCR technique (TaqMan, PE Biosystems) to identify genes that are differentially expressed by human polarised CD4(+) T cell subsets (Th1 or Th2). The goal was to test the feasibility of the detection method in profiling the expression of a set of marker genes important for Th1 and Th2 differentiation. We demonstrate that in polarised human Th1 cells signaling lymphocytic activation molecule (SLAM), a member of the immunoglobulin superfamily, is expressed at 7-25-fold higher levels than in Th2 cells. Along with SLAM, expression of the IL-12 receptor chain beta 2 (IL-12R beta 2) and the IFN-gamma receptor chain beta (IFN-gamma R beta) proved to be useful molecular markers indicating the state of T cell polarisation, as previously reported. Treatment with IL-12 increased SLAM mRNA expression in T cells by 3-4-fold, whereas a number of other cytokines including PDGF-BB, IFN-alpha A, IFN-alpha A/D, IFN-beta, IFN-gamma or IL-9 had no effect. Stimulating T cells by co-ligating CD3 and CD28 increased SLAM protein surface expression in both Th1 and Th2 cells. In conclusion, real-time RT-PCR detection was found to be an accurate, sensitive and highly reproducible method for fast profiling of mRNA expression in Th1 and Th2 cell subsets.

Antigens, CD↗

PubMed1998

Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis.

OBJECTIVE: To assess the persistence of bacterial antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3-triggered reactive arthritis (ReA). METHODS: Peripheral blood samples were obtained from 20 patients with Y. enterocolitica O:3 infection (11 with ReA and 9 without). These samples were studied by immunochemical techniques for the presence of Yersinia antigens at the beginning of infection and up to 4 years thereafter. Synovial fluid samples from 6 of the 11 ReA patients were also studied. RESULTS: The Yersinia antigens lipopolysaccharide and heat-shock protein (HSP) were detected in peripheral blood mononuclear cells and polymorphonuclear phagocytes from all patients studied at the early phase of the disease. They were also found in the synovial fluid cells of patients with Yersinia-triggered ReA. At 4 years after the onset of infection, these bacterial antigens were still detected in the peripheral blood cells of most of the ReA patients studied. CONCLUSION: This study has, for the first time, directly demonstrated that bacterial antigens persist for a long time in patients who develop ReA after Y. enterocolitica O:3 infection. The finding of bacterial HSP in synovial fluid cells could provide a link to the pathogenesis of ReA, since T cell responses of synovial cells have been shown to be directed against that structure. A close similarity between the bacterial and host HSP might contribute to the development of the relatively common, chronic form of this complication.

Antibodies, Bacterial↗

PubMed1998

Invasion by Salmonella typhimurium induces increased expression of the LMP, MECL, and PA28 proteasome genes and changes in the peptide repertoire of HLA-B27.

We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion with Salmonella. The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes. LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2. Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimurium induced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27. We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28alpha and -beta genes, and increased expression of the LMP2 protein by Western blotting. Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion. We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene. This was accompanied by consistent HPLC profile changes for eluted peptides. Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Antigen Presentation↗

PubMed1997

Identification of a Stat-6-responsive element in the promoter of the human interleukin-4 gene.

Interleukin (IL)-4 is an immunomodulatory cytokine produced by a number of cell types including T cells, basophils, and mast cells. This pleiotropic cytokine has a number of immunoregulatory functions; however, the molecular mechanisms controlling the transcription of this gene are not yet completely understood. Several studies have implicated a possible autoregulatory mechanism for its own expression. Here, we have identified a Stat-6-responsive element (Stat-6RE) in the promoter of the human IL-4 gene. Utilizing electrophoretic mobility shift analysis, we have demonstrated the presence of two specific IL-4-responsive DNA-protein complexes in nuclear extracts of both human Th1 and Th2 clones. Phytohemagglutinin-blasted peripheral blood T cells also generated an inducible complex in response to stimulation with IL-4 and the IL-4-like cytokine IL-13. Transient transfection of the murine pre-B cell line BA/F3 stably transfected with the full-length human IL-4 receptor alpha chain demonstrated the ability of multicopy Stat-6RE to initiate transcription from a heterologous promoter upon IL-4 or IL-13 stimulation. These results indicate a possible autocrine mechanism for the regulation of IL-4 gene transcription through the Stat-6RE as well as a possible mechanism for IL-13 regulation of the human IL-4 promoter.

Animals↗

PubMed1996

Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway.

The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.

Base Sequence↗

PubMed1996

CD45 isoforms on human CD4+ T-cell subsets.

We have examined the level of surface expression and functional properties of leukocyte function associated antigen-1, very late antigen-4, and CD45 isoforms on a panel of human CD4+ T-cell clones representative of TH0, TH1, and TH2 cells. There were no qualitative differences in the expression of these antigens among the three types of CD4+ T-cell clones. However, CD45RB was the only CD45 isoform that provided a costimulatory signal in a solid-phase antibody-induced cellular proliferation assay. Additionally, the antigen-induced proliferative response of T-cell clones was inhibited by soluble anti-CD45RO and anti-CD45RB antibodies. Our results suggest that CD45 isoforms differentially provide costimulatory signals to T cells. However, the ability of each CD45 isoform to provide a costimulatory signal does not differ among the TH0, TH1, or TH2 T-cell populations.

CD4-Positive T-Lymphocytes↗

PubMed1995

Modulation of the Grb2-associated protein complex in human CD4+ T cells by receptor activation.

A panel of human CD4+ T cell clones was utilized to dissect and analyze the biochemical consequences of activation of CD3 or CD28. To molecularly characterize receptor-activated proximal signaling events, tyrosine-phosphorylated proteins co-precipitating with a Grb2 fusion protein after receptor activation were analyzed. Ligation of CD28, but not other costimulatory molecules, induced the tyrosine phosphorylation of two previously identified Grb2 binding proteins (pp76 and pp116). A third Grb2 binding protein (pp36) was extensively tyrosine phosphophorylated in response to combined CD3 and CD28 activation, but not in response to ligation of either receptor alone. cAMP and co-ligation of CD45 affected the receptor-activated tyrosine phosphorylation of Grb2-associated proteins. Furthermore, we demonstrated that two signaling molecules, Vav and phosphatidylinositol 3'-kinase (PI(3)K), also interacted with the Grb2 protein complex. The activity of PI(3)K was required for T cell activation, because wortmannin, a PI(3)K inhibitor, blocked T cell proliferation and cytokine production induced by ligation of CD3 and CD28. In conclusion, we demonstrate that in activated human T cell clones, the composition of Grb2 protein complex is modulated by costimulatory signals and cAMP, which may be important for the regulation of intracellular signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Costimulatory signals can selectively modulate cytokine production by subsets of CD4+ T cells.

Analysis of experimental animal models and human clinical samples has indicated that the selective activation of CD4+ T cell subsets with distinct profiles of cytokine production plays an important role in the pathogenesis of human inflammatory and allergic diseases. The possibility that differential activation of costimulatory pathways is a mechanism for selectively modulating cytokine production by CD4+ T cells was tested. The proliferative response and cytokines secreted by a panel of human CD4+ T cell clones, representative of Th1 or Th2/0 cells, in response to activation of different costimulatory pathways was measured. CD28-mediated costimulatory signals induced proliferation and IFN-gamma secretion by Th1 cells. Although CD28-ligation induced Th2/0 cells to proliferate, it did not trigger IL-4 production. Ligation of LFA-1 and CD45 isoforms also generated costimulatory signals activating cytokine secretion by the different types of T cell clones. Th1 cells secreted the same profile of cytokines, irrespective of which costimulatory pathway was engaged. However, the cytokine secreted by a subset of Th2/0 cells varied, depending upon which costimulatory pathways were activated. These results suggest that the costimulatory pathways activated by APCs can selectively influence cytokine production by CD4+ T cell subsets.

Animals↗